The present invention relates to nucleic acid molecules which encode a wheat enzyme involved in starch synthesis in plants. This enzyme is an isoamylase.
The invention furthermore relates to vectors, host cells, plant cells and plants comprising the nucleic add molecules according to the invention.
Furthermore, there are described methods for the generation of transgenic plants which, owing to the introduction of nucleic acid molecules according to the invention, synthesize starch with altered characteristics.
In view of the increasing importance attributed lately to plant constituents as renewable raw materials, one of the objects of biotechnology research addresses the adaptation of these plant raw materials to the needs of the processing industries. Moreover, to allow renewable raw materials to be used in as many fields as possible, a wide diversity of materials must be generated.
Apart from oils, fats and proteins, polysaccharides constitute the important renewable raw materials from plants. Apart from cellulose, starch—which is one of the most important storage substances in higher plants—takes a central position amongst the polysaccharides. In this context, wheat is one of the most important crop plants since it provides approximately 20% of the total starch production in the European Community.
The polysaccharide starch is a polymer of chemically uniform units, the glucose molecules. However, it is a highly complex mixture of different molecule types which differ with regard to their degree of polymerization, the occurrence of branching of the glucose chains and their chain lengths, which, in addition, may be derivatized, for example phosphorylated. Starch therefore does not constitute a uniform raw material. In particular, a distinction is made between amylose starch, an essentially unbranched polymer of alpha-1,4-glycosidically linked glucose molecules, and amylopectin starch, which, in turn, constitutes a complex mixture of glucose chains with various branchings. The branchings occur by the occurrence of additional alpha-1,6-glycosidic linkages. In wheat, amylose starch makes up approximately 11 to 37% of the starch synthesized.
To allow suitable starches to used in the widest possible manner for the widest possible range of industrial needs, it is desirable to provide plants which are capable of synthesizing modified starches which are particularly well suited to various purposes. One possibility of providing such plants is to employ plant-breeding measures. However, since wheat is polyploid in character (tetra- and hexaploid), the exertion of influence by plant breeding proves to be very difficult. A “Waxy” (amylose-free) wheat was generated only recently by crossing naturally occurring mutants (Nakamura et al., Mol. Gen. Genet. 248 (1995), 253-259).
An alternative to plant-breeding methods is the specific modification of starch-producing plants by recomtbinant methods. However, prerequisites are the identification and characterization of the enzymes which are involved in starch synthsis and/or starch modification and the isolation of the nucleic acid molecules encoding these enzymes.
The biochemical pathways which lead to the synthesis of starch are essentially known. Starch synthesis in plant cells takes place in the plastids. In photosynthetically active tissue, these plastids are the chloroplasts and in photosynthetically inactive, starch-storing tissue are amyloplasts.
A further specific alteration of the degree of branching of starch synthesized in plants with the aid of recombinat methods still requires identification of DNA sequences, which encode enzymes involved in starch metabolism, in particular in the introduction or degradation of branching within the starch molecules.
Besides the so-called Q enzymes, which introduce branchings into starch molecules, enzymes occur in plants which are capable of breaking down branchings. These enzymes are called debranching enzymes and, according to their substrate specificity, they are divided into three groups:                (a) The pullulanases, which, in addition to pullulan, also utilize amylopectin as substrate, are found in microorganisms, for example Klebsiella and in plants. In plants, these enzymes are also termed R enzymes.        (b) The isoamylases, which do not utilize pullulan, but indeed glycogen and amylopectin as substrate, are also found in microorganisms and plants. For example, isoamylases have been described in maize (Manners & Carbohydr. Res. 9 (1969), 107) and potato (Ishizaki et al., Agric. Biol. Chem. 47 (1983), 771-779).        (c) The amylo-1,6glucosidases are described in mammals and yeasts and utilize grenzdextrins as substrate.        
In sugar beet, Li et al. (Plant Physiol. 98 (1992), 1277-1284) were only able to find one debranching enzyme of the pullulanase type, in addition to five endoamylases and two exoamylases. This enzyme, which has a size of approx. 100 kD and a pH optimum of 5.5, is localized in the chloroplasts. In spinach, too, a debranching enzyme was described which utilizes pullulan as substrate. The activity both of the spinach debranching enzyme and of the sugar beet debranching enzyme upon reaction with amylopectin as substrate is five times lower in comparison with pullulan as substrat (Ludwig et al., Plant Physiol. 74 (1984), 856-861; Li et al., Plant Physiol. 98 (1992), 1277-1284).
In the agronomically important starch-storing crop plant potato, the activity of a debranching enzyme was studied by Hobson et al. (J. Chem. Soc., (1951), 1451). It was proved successfully that, in contrast to the Q enzyme, this enzyme has no chain-extending activity, but merely hydrolyzes alpha-1,6-glycosidic bonds. However, it has been impossible as yet to characterize the enzyme in greater detail. In the case of potatoes, processes for purifying the debranching enzyme and partial peptide sequences of the purified protein have already been proposed (WO 95/04826). In the case of spinach, the purification of a debranching enzyme and the isolation of suitable cDNA have been described in the meantime (Renz et al., Plant Physiol. 108 (1995), 1342).
In maize, only the existence of one debranching enzyme has been described as yet in the literature. Owing to its substrate specificity, this enzyme is classified as belonging to the group of the isoamylases (see, for example, Hannah et al., Scientia Horticulturae 55 (1993), 177-197 or Garwood (1994) in Starch Chemistry and Technology, Whistler, R. L, BeMiller, J. N., Puschafl, E. F. (eds.), Academic Press San Diego, New York, Boston, 25-86). The corresponding mutant is termed “sugary”. The gene of the sugary locus has been cloned recently (see James et al., Plant C II 7 (1995), 417-429). Apart from the sugary locus, no other gene locus which encodes a protein with debranching enzyme activity is as yet known in maize. Also, there have been no indications to date that other debranching enzyme forms occur in maize. If transgenic maize plants are to be generated which no longer have any debranching enzyme activities whatsoever, for example in order to extend the degree of branching of the amylopectin starch, it is necessary to identify all debranching enzymes forms which occur in maize and to isolate the corresponding genes or cDNA sequences.
To provide further possibilities of altering any starch-storing plant, preferably cereals, in particular wheat, so that it synthesizes a modified starch, it is necessary to identify in each case DNA sequences which encode further isoforms of branching enzymes.